Analysis of the VSX1 Gene in Sporadic Keratoconus Patients from China Tao Guan1†, Xue Wang2†, Li-Bin Zheng3, Hai-Jian Wu1 and Yu-Feng Yao3*
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Guan et al. BMC Ophthalmology (2017) 17:173 DOI 10.1186/s12886-017-0567-3 RESEARCH ARTICLE Open Access Analysis of the VSX1 gene in sporadic keratoconus patients from China Tao Guan1†, Xue Wang2†, Li-Bin Zheng3, Hai-Jian Wu1 and Yu-Feng Yao3* Abstract Background: Keratoconus normally presents as a sporadic disease. Although different studies have found sequence variants of the visual system homeobox 1 (VSX1) gene associated with keratoconus in humans, no research has detected such variants in sporadic keratoconus patients from China. To investigate the possibility of VSX1 being a candidate susceptibility gene for Chinese patients with sporadic keratoconus, we performed sequence screening of this gene in such patients. Methods: Whole DNA was obtained from the leukocytes in the peripheral venous blood of 50 patients with sporadic keratoconus and 50 control subjects without this ocular disorder. Polymerase chain reaction single-strand conformation polymorphism analysis and direct DNA sequencing technology were used to detect sequence variation in the five exons and splicing regions of the introns of the VSX1 gene. The sequencing results were analyzed using DNAstar software. Results: One novel missense heterozygous sequence variant (p.Arg131Pro) was found in the first exon of the VSX1 gene in one keratoconus patient. Another heterozygous sequence variant (p.Gly160Val) in the second exon was found in two keratoconus patients. These variants were not detected in the control subjects. In the third intron of the VSX1 gene, c.8326G > A nucleotide substitution (including heterozygous and homozygous change) was also discovered. The frequency of this variation did not differ significantly between patients and controls, it should belong to single-nucleotide polymorphism of the VSX1 gene. Bioinformatic analysis also predicted that one missense sequence variation (p.Arg131Pro) may not cause a pathogenic change. Conclusions: In this study, we added one novel missense sequence variation (p.Arg131Pro) in the coding region of the VSX1 gene to the range of VSX1 coding region variations observed in patients with sporadic keratoconus from China. Our work suggests that VSX1 sequence variants might be involved in the pathogenesis of sporadic keratoconus, but their precise role in disease causation requires further investigation. Keywords: Sporadic keratoconus, VSX1 gene, Sequence variant, Single-nucleotide polymorphism Background [3, 4]. The pathological features of keratoconus include Keratoconus is a congenital disease involving noninflam- decreased corneal collagen or the abnormal distribution matory corneal ectasia. Its prevalence is estimated to be of collagen fiber, which reduce the mechanical resistance 1/2000 in the general population, and the incidence ratio of the cornea and therefore cause the center of the between males and females is 1.7:1 [1, 2]. Although most cornea to protrude, leading to a thin conical shape. Slit keratoconus cases are sporadic, about 6–10% of cases lamp microscope examinations of keratoconus patients have been reported to have a positive family history. have demonstrated Vogt’s line, Fleischer’s ring, and cor- Keratoconus can exhibit dominant or recessive inherit- neal scarring [5–7]. The onset of the disease occurs during ance; with autosomal dominant inheritance, the disease puberty, involving both eyes, and with highly irregular exhibits variable phenotypes with incomplete penetrance astigmatism. In the late phase, patients show acute corneal edema and scar formation, along with significant vision * Correspondence: [email protected] loss. Although several treatment modalities are available, †Equal contributors 3 severe keratoconus remains an indication for corneal Department of Ophthalmology, Sir Run Run Shaw Hospital, Zhejiang – University School of Medicine, Hangzhou 310016, China transplantation [8 10]. Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Guan et al. BMC Ophthalmology (2017) 17:173 Page 2 of 7 Genome-wide linkage analyses have identified several members and identifying the patient as an isolated case chromosomal loci and genes that may be related to kera- of keratoconus. Another 50 healthy people who under- toconus; some of these were eventually ruled out, while went detailed ocular and ophthalmological examinations for others, an association with this disease could not be that ruled out any ophthalmic diseases were selected as confirmed. One of the candidate genes strongly impli- a control group to determine whether the detected vari- cated in keratoconus is visual system homeobox gene 1 ation was SNP of VSX1 gene. The control subjects in- (VSX1) localized to chromosome 20 p11–q11 [11–14]. cluded 31 males and 19 females, aged from 20 to The VSX1 gene encodes a paired-like homeodomain 36 years old. The age and gender generally matched be- transcription factor. It is associated with eye develop- tween the two groups and systemic physical examina- ment and was first isolated and cloned from a cDNA li- tions were performed in both groups to rule out other brary of the retina of adult goldfish. In the human body, concurrent diseases. This study followed the Declaration the VSX1 gene is expressed in the embryonic craniofa- of Helsinki and was reviewed by the ethics committee of cial region, the granular layer of adult retina, and corneal Taizhou Municipal Hospital. Written informed consent tissue. The human VSX1 gene has five exons and en- was obtained from all of the subjects in this study. codes a 365-amino-acid protein with a homeobox DNA binding domain and a CVC domain, being highly con- Genetic study served among vertebrates. Barbaco et al. [15] reported Three milliliters of peripheral venous blood was ex- that the VSX1 gene was activated in corneal stroma dur- tracted from the subjects and stored in EDTA anticoagu- ing the process of corneal wound repair in vitro and in lation tubes. Genomic DNA was isolated from blood vivo, accompanied by the transformation of corneal stro- leukocytes using the Dzup (Blood) Genomic DNA Isola- mal cells into myofibroblasts. These results implied that tion Reagent (B518205; Sangon Biotech Co., Ltd., Shang- VSX1 gene variants may play an important role in the hai, China), in accordance with the manufacturer’s development of keratoconus. Several variants of the standard methods. VSX1 gene [16–19] have been reported from various Based on previous studies [20, 21] and the DNA se- parts of the world, but a definitive pathogenic role of quence of the human VSX1 gene from the NCBI data- these variants in keratoconus has not yet been estab- base, primers were designed for the five exons of this lished because segregation of these variants was also gene. These primers were synthesized by Sangon Biotech seen in some unaffected individuals. Co., Ltd. (Shanghai, China). Sequences of the primers To our knowledge, this is the first study to perform are listed in Table 1. screening for variants of the VSX1 gene in the Chinese The HBP 220 PCR cycler (HYBAID, England) was population and to explore their role in keratoconus. used for a 50-μL PCR reaction system containing 200 ng Nucleotide variations in the five exons and the splicing of DNA template, 5 μL of 10× buffer solution, regions of introns of the VSX1 gene were examined by 1.5 mmol/L MgCl2, 0.25 μmol/L primers, 100 μmol/L of polymerase chain reaction single-strand conformation each dNTP, and 2 U of Taq DNA polymerase (Sangon polymorphism (PCR-SSCP) analysis and direct DNA se- Biotech Co., Ltd., Shanghai, China). The PCR procedure quencing in 50 Chinese patients with keratoconus, was as follows: 94 °C for 5 min; 35 amplification cycles which were compared with the sequences in 50 controls. of denaturation at 94 °C for 45 s, annealing for 45 s, and extension at 72 °C for 50 s; and finally extension at 72 ° Methods C for 10 min. PCR products were examined by 2% agar- Subjects and clinical examination ose gel electrophoresis and EB (Ethidium Bromide) All sporadic keratoconus patients and control subjects staining. The annealing temperatures of each exon and were identified at the Department of Ophthalmology, the fragment sizes of PCR products are listed in Table 1. Taizhou Municipal Hospital. A total of 50 patients were An 8% (49:1) nondenaturing polyacrylamide gel with diagnosed with sporadic keratoconus during the period 5% glycerol was used for SSCP analysis and silver stain- of March 2013 to July 2015, including 29 males and 21 ing. Here, 5-μL PCR products mixed with sample buffer females, aged from 15 to 35 years old. The diagnosis was (90% formamide, 0.05% bromophenol blue, and 0.05% based on symptoms and the results of slit lamp micro- xylene blue) were processed for denaturation at 95 °C scope examinations; the diagnostic features included for 10 min, placed in an ice bath, and then subjected to progressive curvature of the anterior corneal surface electrophoresis (100 V) at room temperature for 5 to steepening, corneal thinning in the same region, 6 h (when xylene blue reached the bottom of the gel). Fleischer’s ring, and Vogt’s stripes, combined with the Then, the gel was fixed in 10% ethanol for 5 min and corneal surface topography and a caudal surface of the soaked in 1% silver nitrate for 5 min, followed by stain- cornea thicker than 20 μm. Patients were considered as ing in 0.012 mol/L silver nitrate and 0.05% formaldehyde sporadic cases after examining the immediate family for 10 min.